Prior to 1980, the targeting of tumor-bearing sites by radioimmunoglobulin had been demonstrated by a number of laboratories at different institutions (S. E. Order et al., "Use of Isotopic Immunoglobulin in Therapy," Cancer Research 40, 3001-7 (August 1980)). By 1980 it was demonstrated that tumors would concentrate radiolabeled-antibodies to tumor associated antigens and that radiolabeled reagents employed allowed both diagnostic imaging of tumors, e.g., by gamma camera imaging (radioimmunoscintigraphy) and positron tomography, and therapeutic treatment, i.e., reduction in tumor size by the targeting radioactive immunoreagent.
Early targeting with radiolabeled immunoreagents was carried out with radioactive-iodine. However, as noted by Scheinberg et al, "Tumor Imaging with Radioactive Metal Chelates Conjugated to Monoclonal Antibodies," Science 215, No. 19, 1511-13 (March 1982 ), iodine isotopes pose several problems, particularly with respect to scanning of tumor images. Of the three commonly available isotopic forms, only .sup.123 I has the appropriate emission characteristics for imaging and a short enough half-life to be safely used diagnostically. The gamma radiation of .sup.125 I is too weak for imaging. .sup.131 I has often been used but is undesirable because of its long half-life and high energy gamma and cytotoxic beta radiations. .sup.131 I has also been used therapeutically for large tumors, but appears ineffective in the treatment of small tumors. Moreover, rapid metabolism of radioiodinated antibodies allows incorporation of the iodine into the thyroid and active excretion of the iodine by the stomach and urinary tract. This dispersion of the radioactive iodine hinders imaging of specific tumors since the tumors are hidden by background radiation.
In addition to tumor targeting with radioactive antibodies for diagnostic imaging and therapeutic treatment, similar targeting has been accomplished for diagnostic imaging of infarcts, specifically, myocardial infarcts, using antibodies to canine cardiac myosin (Khaw et al, "Myocardial Infarct Imaging of Antibodies to Canine Cardiac Myosin Indium-111- Diethylenetriamine Pentaacetic Acid," Science 209, 295-7 (July 1980), and for imaging atherosclerosis by targeting atherosclerotic plaques. The same disadvantages in the use of radioactive iodine exist for diagnostic infarct imaging as for tumor imaging and therapeutic treatment.
It is known that .sup.111 In can be complexed with polyaminocarboxylic acids such as ethylene diaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). However, the covalent linkage of proteins (antibodies) to these complexing agents, accomplished by acylation with activated carbonyls, aromatic diazonium coupling, or bromoacetylation is inefficient, even though the isocyanatobenzyl derivatives described by Brechbiel et al "Synthesis of 1-(p-Isothiocyanatobenzyl)Derivatives of DTPA and EDTA. Antibody Labeling and Tumor Imaging Studies," Inorg. Chem. 25, 2772-81 (1986)) were created to facilitate covalent attachment of proteins with the complexing agents.
Recently, research efforts have been directed to improved antibodies (Ab's), e.g., monoclonal, specific antibodies for specific targeting, antibodies that complex or bind directly with radionuclides, preferred radionuclides and combinations thereof with antibodies and complexing agents. Some attempts have been made towards improving complexing agents.
Nonetheless, EDTA and especially DTPA and derivatives thereof have remained the prevalent complexing agents to covalently bind antibody and coordinately complex metallic radionuclides. However, the inadequacies of DTPA have been noted, for example, by Parker et al, "Implementation of Macrocycle Conjugated Antibodies for Tumor Targeting," Pure and Appl. Chem., 61, No. 9, 1637-41 (1989) . . . "Conventionally the metal radionuclide has been complexed by an acyclic chelate (e.g. EDTA or DTPA) which is covalently linked to the antibody. None of the chelates is adequate because the metal tends to dissociate in vivo, . . . " and by Cox et al, "Synthesis of a Kinetically Stable Yttrium-90 Labelled Macrocycle-Antibody Conjugate," J. Chem. Soc., Chem. Commun. 797-8 (1989) . . . "Yttrium-90 is an attractive isotope for therapy . . . but its clinical use will be very limited because of bone marrow toxicity, resulting from acid-promoted release of .sup.90 y from an antibody linked chelate such as diethylenetriamine-pentaacetic (DTPA)."
The attempts to develop improved complexing agents have provided materials which have their shortcomings. For example, Craig et al "Towards Tumor Imaging with Indium-111 Labelled Macrocycle-Antibody Conjugates," J. Chem. Soc. Chem. Commune, 794-6 (1989) describe macrocyclic hexacoordinating ligands but state that "The limiting feature of this approach is that .sup.111 In labelling of the macrocycle is required before antibody conjugation. Indium binding by (4) is insufficiently fast at 37.degree. C. for efficient radiolabeling . . . Other tribasic triazamacrocyclic ligands were screened therefore for their ability to bind indium rapidly under mild conditions (20.degree. C., pH 5, &lt;1 h), yet still form a kinetically stable complex in vivo. . . However, only (6) proved effective when the ligand concentration was 10 .mu.M, and under these conditions a 96% radiolabeling yield was determined (30 rain, pH 5, 20.degree. C.)."
Nevertheless, thirty minutes is still unsatisfactory. It would be highly desirable to have complexing agents superior to EDTA and DTPA which would coordinately bind preferred radionuclides such as In, Y, Sc, Ga, Ge, etc. within a few minutes, i.e., in less than about 5 min, immediately prior to administration of the reagent to the patient, especially when a short-lived radionuclide must necessarily be generated from a longer-lived radionuclide at the time of treatment of the patient.
It should be noted that complexes of yttrium, a preferred radionuclide, tend to be less stable than those of indium (Mather et al, "Labelling Monoclonal Antibodies with Yttrium 90," Eur. J. Nucl. Med. 15, 307-312 (1989)) with respect to conventional complexes. Mather et al teach that biodistribution studies in cancer patients using radiolabeled antibodies have suggested that the in vivo stability of yttrium-labeled antibodies is not as great as their .sup.111 In-labelled counterparts and that these findings are supported by other recent publications in the field.
When chelating agents are covalently bonded to proteins (Ab's), the proteins usually are capable of accepting far more than one molecule of the chelating agent because they contain a host of amine and sulfhydryl groups through which the chelating agents are bound. It is often very important to determine how many chelating sites are bound to each protein molecule. The most convenient way to accomplish this is by spectrophotometric means. However, prior art chelating agents and chelates thereof have spectra that overlap with those of useful proteins, and an analytical determination of the number of chelating or chelated sites per molecule of protein cannot be made by spectroscopy since the overlapping spectra mask each other. It would thus be highly desirable to obtain chelating agents for conjugation to proteins whose spectra, and whose metal chelate spectra, do not overlap with that of the proteins to which the chelating agents are chemically bonded.
Another problem with some prior art compositions is that the chelator must be activated by a reducing agent before forming the radionuclide chelate. If the protein conjugates are to be formed prior to formation of the radionuclide chelate, then the reducing agent employed for activating the complexing agent can degrade the protein. For example, the preferred chelating agents currently used for complexing technetium (Tc) and rhenium (Re) complex to the metals via sulfur-containing groups which must be reduced with a reducing agent (dithiothreitol) to activate the chelator before forming the radionuclide chelate. If the protein conjugate containing disulfide bonds is formed prior to reduction, then the reducing agent can degrade the protein. It would be highly desirable to have chelating agents capable of forming conjugates with proteins before complexing with radionuclides, and particularly chelating agents for Tc and Re which do not require an activation step involving a reducing agent prior to complexation.
In summary, the various currently available radiolabeled antibodies and chelating agents employed for making immunoreactive conjugates by covalently bonding of a chelating agent to the immunoreactive protein, and radionuclide complexes thereof for use in diagnostic imaging and targeted therapeutics suffer from certain of the following disadvantages: 1) toxicity; 2) dispersion of the reagent due to rapid metabolism; 3) inadequate emission characteristics; 4) inefficient covalent bonding with protein for conjugate preparation; 5) slow complexation with metals; 6) unstable metal complexation, e.g., with respect to temperature, time or pH; 7) inability to form conjugates and store until metal complexation is desired; 8) inability to spectrophotometrically analyze the radionuclide complex reagent; and 9) inability to complex without activation steps that degrade protein.